WO2021120019A1 - Composition d'immunisation et son procédé de préparation - Google Patents

Composition d'immunisation et son procédé de préparation Download PDF

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Publication number
WO2021120019A1
WO2021120019A1 PCT/CN2019/126096 CN2019126096W WO2021120019A1 WO 2021120019 A1 WO2021120019 A1 WO 2021120019A1 CN 2019126096 W CN2019126096 W CN 2019126096W WO 2021120019 A1 WO2021120019 A1 WO 2021120019A1
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amino acid
polypeptide
seq
kpn
composition
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PCT/CN2019/126096
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English (en)
Chinese (zh)
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张宝中
黄建东
胡丹玉
窦颖
胡景初
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中国科学院深圳先进技术研究院
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Publication of WO2021120019A1 publication Critical patent/WO2021120019A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/26—Klebsiella (G)

Definitions

  • the present invention relates to the field of medical immunity, in particular to a composition for immunization and a preparation method thereof.
  • Klebsiella pneumoniae (Klebsiella pneumoniae) is a Gram-negative Brevibacterium with a thicker capsule, often arranged in pairs end-to-end. It is one of the important conditional pathogens and iatrogenic infections. . Klebsiella exists in the upper respiratory tract and intestinal tract of humans. It is a genus of bacteria in the human body itself. When the body's immunity is reduced, it enters the lungs through the respiratory tract to cause infection. Klebsiella pneumoniae can cause pneumonia, bronchitis, urinary tract and trauma infections, and sometimes serious diseases such as sepsis, meningitis and peritonitis.
  • Klebsiella pneumoniae with different drug-resistant phenotypes such as extended-spectrum ⁇ -lactamase-resistant Klebsiella pneumoniae, carbapenemase-resistant Klebsiella pneumoniae and multi-drug-resistant Klebsiella pneumoniae Epidemic spread occurs worldwide, causing hospital infection rates to increase year by year, and the continuous increase of multi-drug resistant strains often leads to the failure of clinical antimicrobial treatment and the prolonged course of the disease.
  • the available clinical options for antimicrobial drugs are extremely limited.
  • Klebsiella pneumonia type III fimbriae structural gene A there are 8 B cell dominant epitope regions that can be used to prepare monoclonal antibodies and design epitope vaccines.
  • rkA fimbriae structural gene A
  • a variety of new bacterial vaccines with different compositions have been developed in recent years, including polysaccharide vaccines, polysaccharide conjugate vaccines, protein vaccines and microcapsule vaccines.
  • the 24-valent polysaccharide vaccine of Klebsiella pneumoniae is the most promising method, and it has passed the phase I clinical trial.
  • the protection provided by the 24-valent vaccine can only cover no more than 70% of the strains, and due to the complicated production process, the researchers did not continue this study after the phase I clinical trial.
  • the main types of vaccines for Klebsiella pneumoniae currently on the market and more researched are polysaccharides, lipopolysaccharides, and proteins. These vaccines all have good immunogenicity and antigenicity, but each has its own shortcomings.
  • the capsular polysaccharide in Klebsiella pneumoniae is located in the outermost layer of the cell and is an important virulence factor.
  • the monovalent capsular polysaccharide vaccine cannot provide protection against Klebsiella pneumoniae.
  • Conjugated vaccines are the use of chemical methods to covalently bind polysaccharide antigens to carrier proteins to form glycoproteins, so as to achieve a lasting immune protection effect. Some authors have constructed a conjugate vaccine combining O-antigen and outer membrane protein, and a series of experiments have proved that this conjugate vaccine has lasting immunogenicity. Due to the single type of carrier protein and the high production cost, the development of conjugate vaccines is limited to a certain extent.
  • the main technical problem solved by the present invention is to provide a composition for immunization and a preparation method thereof, which can resist Klebsiella pneumoniae infection.
  • the technical solution adopted by the present invention is to provide a composition for immunization, including:
  • the composition includes at least two polypeptides, each of which is different, and both are outer membrane polypeptides used for immunizing various Klebsiella pneumoniae; wherein, the polypeptides include the first polypeptide, the second polypeptide, and the third polypeptide.
  • Polypeptide, fourth polypeptide, fifth polypeptide, and sixth polypeptide are examples of polypeptides, and amino acids that are amino acids that are amino acids that are amino acids used for immunizing various Klebsiella pneumoniae; wherein, the polypeptides include the first polypeptide, the second polypeptide, and the third polypeptide.
  • Polypeptide, fourth polypeptide, fifth polypeptide, and sixth polypeptide are polypeptides, and both are outer membrane polypeptides used for immunizing various Klebsiella pneumoniae; wherein, the polypeptides include the first polypeptide, the second polypeptide, and the third polypeptide.
  • the first polypeptide includes at least 70% of the amino acid sequence in SEQ ID1 and/or includes at least 7 contiguous amino acid fragments in SEQ ID1; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID1;
  • the second polypeptide includes at least 70% of the amino acid sequence in SEQ ID2 and/or includes at least 7 contiguous amino acid fragments in SEQ ID2; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID2;
  • the third polypeptide includes at least 70% of the amino acid sequence in SEQ ID3 and/or includes at least 7 contiguous amino acid fragments in SEQ ID3; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID3;
  • the fourth polypeptide includes at least 70% of the amino acid sequence in SEQ ID4 and/or includes at least 7 contiguous amino acid fragments in SEQ ID4; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID4;
  • the fifth polypeptide includes at least 70% of the amino acid sequence in SEQ ID5 and/or includes at least 7 contiguous amino acid fragments in SEQ ID5; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID5;
  • the sixth polypeptide includes at least 70% of the amino acid sequence in SEQ ID6 and/or includes at least 7 contiguous amino acid fragments in SEQ ID6; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID6.
  • the composition includes histidine buffer.
  • composition further includes at least one of Klebsiella pneumoniae capsular polysaccharide, lipopolysaccharide and carrier protein.
  • the coupling structure of the composition is NH 2 -A- ⁇ -XL- ⁇ NB-COOH; where A represents an optional N-terminal amino acid sequence; B represents an optional C-terminal amino acid sequence; L represents Optionally at least one polypeptide.
  • composition further includes Klebsiella non-pneumoniae antigen.
  • the polypeptide is prepared by recombinant expression in a heterologous host; wherein, the heterologous host is a prokaryotic or eukaryotic organism.
  • the technical solution adopted by the present invention is to provide a method for preparing a composition for immunization, including:
  • the polypeptides are outer membrane polypeptides used for immunization of various Klebsiella pneumoniae; the obtained polypeptides are mixed and prepared into antigens.
  • the present invention has identified six kinds of Klebsiella pneumoniae outer membrane polypeptides that can be used for immunization alone or in combination. By combining different polypeptides, they can form a broad-spectrum protection across different serums.
  • the Klebsiella pneumoniae vaccine composition can effectively resist Klebsiella pneumoniae infection.
  • Figure 1 is a schematic diagram of the process of preparing six polypeptides in an embodiment of the present invention
  • Figure 2 is a schematic diagram of the egg/IgG1/IgG2a antibody titer levels in mouse serum caused by six polypeptides in Example 1 of the present invention
  • Figure 3 is a schematic diagram of the survival rate of mice immunized with antigen in Example 2 of the present invention after being infected with a lethal dose of bacteremia model;
  • Example 4 is a schematic diagram of the survival rate of mice immunized with antigen in Example 3 of the present invention after infection with a lethal dose of pneumonia model;
  • Example 5 is a schematic diagram of the bacterial load in different tissues of the mice immunized with the antigen in Example 4 of the present invention after being infected with a non-lethal dose of pneumonia model;
  • FIG. 6 is a statistical schematic diagram of cytokines secreted by T cells induced by the spleen tissue of the antigen-immunized mouse in Example 5 of the present invention under the synchronous stimulation of the antigen;
  • Fig. 7 is a diagram showing the effect of serum conditioning HL60 cells of mice immunized with antigen in Example 6 of the present invention to promote their phagocytosis of Klebsiella pneumoniae.
  • first, second, third, etc. may be used to describe XXX in the embodiments of the present invention, these XXX should not be limited to these terms. These terms are only used to distinguish XXX from each other.
  • the first XXX may also be referred to as the second XXX, and similarly, the second XXX may also be referred to as the first XXX.
  • the present invention identifies six kinds of Klebsiella pneumoniae outer membrane polypeptides that can be used alone or in combination for immunization, namely: the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, and the first polypeptide. Five peptides and sixth peptides.
  • the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide, and the sixth polypeptide correspond to Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp003 polypeptide, Kpn_Omp004 polypeptide, Kpn_Omp005 polypeptide, and Kpn_Omp006 polypeptide, respectively.
  • Kpn_Omp001 antigen, Kpn_Omp002 antigen, Kpn_Omp003 antigen, Kpn_Omp004 antigen, Kpn_Omp005 antigen and Kpn_Omp006 antigen used below are equivalent to the terms "Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp003 polypeptide, Kpn_005Kp006 polypeptide, and polypeptide”.
  • Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, Kpn_Omp005 and Kpn_Omp006 used below are equivalent to the terms "Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp003 polypeptide, Kpn_Omp006 and polypeptide".
  • the identified polypeptides of the present invention include certain amino acid sequences, which are 100% identical or greater than or equal to 70% of a certain polypeptide sequence disclosed in the sequence list.
  • the identified polypeptide has at least 70% sequence identity with the reference amino acid sequence. Most typically, when such a polypeptide is only a few adjacent amino acid fragments in the corresponding amino acid sequence, the polypeptide and the reference amino acid sequence A certain polypeptide sequence disclosed has 100% sequence identity.
  • sequence list is a list of amino acid sequences corresponding to the six polypeptides identified in the present invention.
  • the first polypeptide includes at least 70% of the amino acid sequence in SEQ ID1 and/or includes at least 7 contiguous amino acid fragments in SEQ ID1; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID1;
  • the second polypeptide includes at least 70% of the amino acid sequence in SEQ ID2 and/or includes at least 7 contiguous amino acid fragments in SEQ ID2; wherein the amino acid fragment includes the amino acid sequence of the epitope in SEQ ID2;
  • the third polypeptide includes at least 70% of the amino acid sequence in SEQ ID3 and/or includes at least 7 contiguous amino acid fragments in SEQ ID3; wherein the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID3;
  • the fourth polypeptide includes at least 70% of the amino acid sequence in SEQ ID4 and/or includes at least 7 contiguous amino acid fragments in SEQ ID4; wherein the amino acid fragment includes the amino acid sequence of the epitope in SEQ ID4;
  • the sixth polypeptide includes at least 70% of the amino acid sequence in SEQ ID 6 and/or includes at least 7 contiguous amino acid fragments in SEQ ID 6; wherein, the amino acid fragment includes the amino acid sequence of the antigen epitope in SEQ ID 6.
  • the composition includes at least two of Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp003 polypeptide, Kpn_Omp004 polypeptide, Kpn_Omp005 polypeptide, and Kpn_Omp006 polypeptide, capable of eliciting an immune response and providing against different types of Klebsiella pneumoniae Protective immunity.
  • the Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, and Kpn_Omp005 polypeptide among the six polypeptides constitute a polypeptide subgroup, and the composition includes at least two polypeptides selected from the polypeptide subgroup.
  • composition provided by the present invention includes at least two polypeptides in the polypeptide subgroup.
  • the polypeptide is adsorbed on the adjuvant.
  • the present invention uses aluminum hydroxide adjuvant to adsorb the polypeptide in the composition.
  • a combination of adjuvants that activate Th1 and/or Th2 cellular immunity such as CPG and alum, are used.
  • Histidine buffer is included in any of the above compositions.
  • polypeptides identified in the present invention can be present in the composition as separate polypeptide components, or can be fused into longer peptide chains. If the composition includes at least two polypeptides, the combination of polypeptides may be present in the composition in the form of fusion polypeptides.
  • the fusion polypeptide may include at least one polypeptide sequence among Kpn_Omp001 polypeptide, Kpn_Omp002 polypeptide, Kpn_Omp003 polypeptide, Kpn_Omp004 polypeptide, Kpn_Omp005 polypeptide, and Kpn_Omp006 polypeptide.
  • the fusion polypeptide may contain two or more variants of the same polypeptide when there are partial changes between strains. Different fusion polypeptides can be mixed in one formulation.
  • the composition of the present invention can also be fused with another protein.
  • the composition further includes at least one of Klebsiella pneumoniae capsular polysaccharide (K antigen), lipopolysaccharide (O antigen), and carrier protein.
  • K antigen Klebsiella pneumoniae capsular polysaccharide
  • O antigen lipopolysaccharide
  • carrier protein carrier protein
  • composition of the present invention also includes a Klebsiella non-pneumonia antigen.
  • polypeptides identified in the present invention can be combined with antigens of bacteria associated with hospital infections, and the resulting composition can provide broad-spectrum protective immunity.
  • the immunogenic polypeptides provided by the present invention can be selected from natural polypeptides, fusion polypeptides, glycosylated polypeptides, non-glycosylated polypeptides, lipidated polypeptides, non-esterified polypeptides, phosphorylated polypeptides, and non-phosphorylated polypeptides. , Myristoylated polypeptides, non-myristoylated polypeptides, monomers, multimers, particles, and denatured polypeptides.
  • the immunogenic polypeptide provided by the present invention is prepared by recombinant expression in a heterologous host.
  • the heterologous host can be a prokaryotic or eukaryotic organism.
  • the sequence encoding the polypeptide is operably linked to a promoter in the vector.
  • operably linked means that the nucleotide sequence is linked to the promoter in a manner that allows the nucleotide sequence to be expressed.
  • Figure 1 is a schematic diagram of a process for preparing six polypeptides in an embodiment of the present invention. The method includes the following steps:
  • this step first explore the various conditions in the process of enzymatic treatment of Klebsiella pneumoniae, and complete the "shaving" of its viable bacteria.
  • the growth stage of the bacteria, the choice of enzyme and reaction system, the combination of different enzymes, the reaction temperature, and the reaction time will all affect the digestion effect.
  • the present invention tests trypsin, proteinase K, chymotrypsin and pepsin, and selects the optimal enzyme combination according to the effect of enzyme digestion.
  • a false positive control group was set up to eliminate the pollution caused by spontaneous lysis, and then the bacterial surface protein was enriched by multiple rounds of enzyme digestion, and the enriched bacterial surface protein was subjected to liquid phase mass spectrometry (LC-MS/MS) to obtain a relatively complete Surface proteome, and then search for the identification results in the Klebsiella pneumoniae protein database and analyze cross-comparisons for mutual verification, complete the further classification of potential targets, and then screen potential antigen targets based on protein abundance and conservation .
  • LC-MS/MS liquid phase mass spectrometry
  • the polymerase chain reaction is amplified into the antigen gene fragment (containing NcoI and XhoI sites) and the commercial vector pET28a.
  • Recombinant plasmid was obtained by double digestion with NcoI and XhoI and ligation with T4DNA ligase.
  • the recombinant plasmid was transformed into E. coli BL-21 strain, and the strain was spread on the surface of a kanamycin-resistant plate and placed in a 37°C incubator to grow overnight. Pick a single clone to kanamycin-resistant LB (broth medium) medium, and place it in a shaker at 37° C. and 200 rpm for overnight culture.
  • the embodiment of the present invention provides a composition for immunization, which can cause cell-mediated immune response and humoral immune response, induce cell-mediated immunity and long-acting antibodies, so as to be in contact with Klebsiella pneumoniae reacts quickly.
  • Two types of T cells, CD4 and CD8 cells are necessary to initiate and/or enhance cell-mediated immunity and humoral immunity.
  • CD8 T cells can express CD8 co-receptors and are called cytotoxic T lymphocytes (CTL).
  • CD8 T cells can recognize antigenic peptides on MHC type I molecules.
  • CD4 T cells can express CD4 co-receptors and are called T cell helper cells.
  • CD4T cells can recognize antigen peptides on MHC type II molecules. After the antigen peptide interacts with MHC type II molecules, CD4 cells can secrete cytokines, and these secreted cytokines can activate B cells, cytotoxic T cells, macrophages and other cells involved in the immune response.
  • the goal of this example is to explore the immunogenicity and protective efficacy of six recombinant outer membrane polypeptides injected subcutaneously into the neck in a mouse model.
  • the antigen doses of 25ug Kpn_Omp001, 25ug Kpn_Omp002, 25ug Kpn_Omp003, 25ug Kpn_Omp004, 25ug Kpn_Omp005 and 25ug Kpn_Omp were used to study vaccines based on Klebsiella pneumoniae.
  • Vaccine 1 25ug Kpn_Omp001 antigen, aluminum hydroxide adjuvant, which contains histidine buffer; wherein the final concentration of aluminum hydroxide adjuvant in the vaccine composition is 1 mg/ml (10%).
  • Vaccine 3 25ug Kpn_Omp003 antigen, aluminum hydroxide adjuvant, which contains histidine buffer; wherein the final concentration of aluminum hydroxide adjuvant in the vaccine composition is 1 mg/ml (10%).
  • Vaccine 4 25ug Kpn_Omp004 antigen, aluminum hydroxide adjuvant, which contains histidine buffer; wherein the final concentration of aluminum hydroxide adjuvant in the vaccine composition is 1 mg/ml (10%).
  • Vaccine 5 25ug Kpn_Omp005 antigen, aluminum hydroxide adjuvant, which contains histidine buffer; wherein the final concentration of aluminum hydroxide adjuvant in the vaccine composition is 1 mg/ml (10%).
  • mice in 6 groups received three subcutaneous injections of 25ug antigen protein in the neck on the 0th, 14th, and 28th days.
  • the total volume of each animal was 100ul, and aluminum hydroxide adjuvant accounted for 10%.
  • Enzyme-linked immunosorbent assay was used to determine whether Kpn_Omp001 antigen, Kpn_Omp002 antigen, Kpn_Omp003 antigen, Kpn_Omp004 antigen, Kpn_Omp005 antigen, and Kpn_Omp006 antigen can be induced
  • ELISA Enzyme-linked immunosorbent assay
  • Figure 2 is a schematic diagram of the titers of IgG/IgG1/IgG2a antibodies in mouse serum caused by the six polypeptides in Example 1 of the present invention.
  • the titer levels of IgG/IgG1/IgG2a antibodies in mouse serum are as follows:
  • mice vaccinated with 6 kinds of outer membrane antigens can activate the level of humoral immune response after three injections of immune protein.
  • the goal of this example is to explore the protective effect of Klebsiella pneumoniae antigen on a mouse model of bacteremia model infected by tail vein injection.
  • mice that were immunized with protein three times in Example 1 were injected with K. pneumoniae 260 (bacteremia model) at a dose of 1 ⁇ 109 CFU through the tail vein, and the health of the infected mice was monitored daily for two weeks.
  • K. pneumoniae 260 bacteremia model
  • FIG. 3 is a schematic diagram of the survival rate of the mice immunized with the antigen after being infected with a lethal dose of bacteremia model in Example 2 of the present invention.
  • the survival situation is as follows:
  • the mortality rate of the mice within three days after the infection of the bacteremia model was 20%, and the mortality rate remained unchanged for more than ten days thereafter.
  • the survival rate of the mice immunized with the Kpn_Omp001 antigen was 80% after two weeks.
  • mice within three days after infection with the bacteremia model was 30%, the mortality rate of mice within two weeks was 50%, and the survival rate of mice immunized with Kpn_Omp002 antigen was 50% after two weeks.
  • the mortality rate of the mice within three days after the infection of the bacteremia model was 70%, and the mortality rate remained unchanged for more than ten days thereafter.
  • the survival rate of the mice immunized with the Kpn_Omp002 antigen was 30% after two weeks.
  • the mortality rate of the mice within three days after the infection of the bacteremia model was 80%, and the mortality rate remained unchanged for more than ten days thereafter.
  • the survival rate of the mice immunized with the Kpn_Omp002 antigen was 20% after two weeks.
  • the mortality rate of the mice within three days after the infection of the bacteremia model was 50%, and the mortality rate remained unchanged for more than ten days thereafter.
  • the survival rate of the mice immunized with the Kpn_Omp002 antigen was 50% after two weeks.
  • mice within three days after infection with the bacteremia model was 80%, and within two weeks, the mortality rate was 100%.
  • mice After immunization with Kpn_Omp003 antigen and Kpn_Omp004 antigen, the antibody level in mice was not enough to protect the mice. After being infected with a lethal dose of bacteremia model, the survival rate of mice within two weeks was low; After Kpn_Omp001 antigen, Kpn_Omp002 antigen and Kpn_Omp005 antigen, the level of antibody in mice protects mice higher. After infection with lethal dose of bacteremia model, the survival rate of mice is 80%, 50% and 50%, respectively .
  • the goal of this example is to explore the protective effect of Klebsiella pneumoniae antigen on a mouse model of pneumonia infected by intranasal injection.
  • mice were anesthetized with pentobarbital sodium, and K.pneumonia 260 (pneumonia model) (25 ⁇ L in each nostril) was injected intranasally with a dose of 1 ⁇ 109 CFU to monitor the health of the infected mice The condition lasted for 200 hours.
  • FIG. 4 is the survival rate of the mice immunized with the antigen in Example 3 of the present invention after being infected with a lethal dose of pneumonia model.
  • the survival situation is as follows:
  • mice within 96 hours after infection with the pneumonia model was 60%, and the mortality rate within more than 100 hours thereafter was 80%.
  • the survival rate of mice immunized with Kpn_Omp002 antigen was 20% after 200 hours.
  • mice within 96 hours after infection with the pneumonia model was 70%, and the mortality rate remained unchanged for more than 100 hours thereafter.
  • the survival rate of mice immunized with Kpn_Omp005 antigen after 200 hours was 30%.
  • the mortality rate of the mice was 100% within 96 hours after being infected with the pneumonia model.
  • the objective of this example is to explore the changes in bacterial load in different tissues of mice after an intravenous injection of a non-lethal dose of a pneumonia model in a mouse model immunized with an antigen.
  • K.pneumonia260 (pneumonia model) was injected intravenously with a dose of 5 ⁇ 108 CFU, and the health of the infected mice was monitored.
  • the mice were anesthetized with pentobarbital sodium and bloodletted Perform euthanasia.
  • the spleen, kidney and lung tissues of the mice were collected under aseptic conditions. Different tissue samples were serially diluted on the BHI plate and the number of CFU of K. pneumonia 260 was counted.
  • FIG. 5 is a schematic diagram of the bacterial load in different tissues of a pneumonia model of a non-lethal dose of a pneumonia model in mice immunized with an antigen in Example 4 of the present invention.
  • the goal of this example is to explore the specific situation of inducing T cells to secrete cytokines in the tissues of mice immunized with antigens.
  • mice 72 hours after the mice received the third booster immunization, the mice were anesthetized with sodium pentobarbital and euthanized by bleeding.
  • the mouse spleen tissue was collected under aseptic conditions, and after thorough grinding, splenic mononuclear cells were separated using lymphocyte separation solution.
  • FIG. 6 is a statistical schematic diagram of the cytokines secreted by T cells induced by the spleen tissue of the antigen-immunized mouse in Example 5 of the present invention under the synchronous stimulation of the antigen.
  • mice immunized with Kpn_Omp001 antigen, Kpn_Omp002 antigen and Kpn_Omp005 antigen can induce cellular immune responses of Th1, Th2 and Th17 related to IFN- ⁇ , IL-4 and IL17a.
  • the goal of this example is to explore whether the serum of mice immunized with antigen can regulate cells to promote the phagocytosis of Klebsiella pneumoniae.
  • the mice received the third booster immunization blood was taken and the serum was separated to prepare a sample, and the resulting serum sample was the antiserum.
  • the antiserum samples were incubated with K. pneumonia 260 and differentiated HL60 cells to detect and analyze the phagocytosis of K. pneumoniae by HL60 cells.
  • the serum of unimmunized mice was incubated with K. pneumonia 260 and differentiated HL60 cells, and the phagocytosis of HL60 cells on Klebsiella pneumoniae was detected and analyzed.
  • FIG. 7 is a diagram showing the effect of serum conditioning HL60 cells of mice immunized with antigen in Example 6 of the present invention to promote their phagocytosis of Klebsiella pneumoniae.
  • mice immunized with Kpn_Omp001, Kpn_Omp002, Kpn_Omp005 and Kpn_Omp006 antigens was extracted to prepare antiserum samples, which were then combined with K.pneumonia 260 and differentiated HL60 cells. After incubating together, HL-60 cells can kill more than 25% of Klebsiella pneumoniae.

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Abstract

L'invention concerne une composition pour l'immunisation et son procédé de préparation. La composition comprend au moins deux polypeptides qui sont différents l'un de l'autre et sont des polypeptides de membrane externe pour une immunisation contre divers types de Klebsiella pneumoniae, les polypeptides comprenant un premier polypeptide, un deuxième polypeptide, un troisième polypeptide, un quatrième polypeptide, un cinquième polypeptide et un sixième polypeptide.
PCT/CN2019/126096 2019-12-17 2019-12-17 Composition d'immunisation et son procédé de préparation WO2021120019A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040518A1 (fr) * 2000-11-17 2002-05-23 Pierre Fabre Medicament Domaine periplasmique d'une proteine omp d'enterobacterie et son utilisation comme porteur ou adjuvant
FR2828106A1 (fr) * 2001-08-02 2003-02-07 Pf Medicament Utilisation d'une omp d'enterobacterie de faible masse moleculaire comme porteur et/ou adjuvant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040518A1 (fr) * 2000-11-17 2002-05-23 Pierre Fabre Medicament Domaine periplasmique d'une proteine omp d'enterobacterie et son utilisation comme porteur ou adjuvant
FR2828106A1 (fr) * 2001-08-02 2003-02-07 Pf Medicament Utilisation d'une omp d'enterobacterie de faible masse moleculaire comme porteur et/ou adjuvant

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